The technical field of this invention concerns internal controls for immunoassay devices and methods for the detection of an analyte in a liquid sample. More particularly, the present invention relates to the use of non-antibody control substances, such as Protein A, which are capable of binding to the Fc fragment of immunoglobulin. These substances serve as internal controls in such assay devices and methods, to ensure that reagents used in the assay are functioning and that the assay is performed properly.
Typically in an immunoassay for the detection of an antibody specific to an antigen (hereinafter referred to as "analyte antibody"), such as a test for the detection of antibodies to human immunodeficiency virus (HIV) or hepatitis C virus (HCV) antigen, a sample suspected of containing the analyte antibody is added to an immunoassay device surface having the particular antigen immobilized thereon. After the sample is added, an antibody specific to human antibody which has been labeled with an enzyme, a metal colloid, or some other marker, is added. If analyte antibody from the patient sample has bound to the antigen, the labeled antibody will also bind, thus indicating a positive test result.
Because immunoassays are often performed by laboratory personnel, human error is possible. Therefore, it is necessary to have controls to ensure that the testing is performed properly. Also because the reagents used in these immunoassays can often be of limited stability, controls are necessary to ensure that the reagents are functioning properly. Typically, when testing for the presence of analyte antibody, the immunoassay device will have a built-in control of an antibody directed to human immunoglobulin G (IgG), IgM, IgE, or IgA. Thus when a patient sample (typically plasma or serum) is added to the immunoassay device, immunoglobulin will bind to the internal control regardless of whether there is analyte antibody present in the sample. When labeled antibody is added, presence of the label at both internal control and antigen locations indicates the presence of the analyte antibody in the tested sample. Presence of the label at the internal control but not at the antigen location, indicates a negative test result. If little or no label is present at the internal control, then there was an error in the assay procedure, or one or more of the reagents was non-functioning, or the sample was diluted beyond the sensitivity of the system to detect immunoglobulin.
In an immunoassay for the detection of a specific antigen in a patient sample (hereinafter referred to as "analyte antigen"), an internal control will typically comprise the same antigen as the antigen to be detected. When a patient sample is added to the immunoassay device, any analyte antigen within the sample will bind to the area on the immunoassay device that has capture antibody immobilized thereon that is specific to the analyte antigen. When labeled antibody specific to the analyte antigen is added, it will bind to any analyte antigen captured from the patient sample and it will bind to the antigen of the internal control. This controls for the proper addition and functioning of the labeled antibody, but does not control for the proper addition of sample because if sample were inadvertently omitted from the immunoassay procedure, the internal control would still be able to bind to the labeled-antibody.
Protein A from Staphylococcus aureus has the ability to bind to the C(H2) and C(H3) domains of the Fc region of immunoglobulins, particularly IgG. In addition to this dominant Fc interaction which is a pseudo-immuno (non-antibody type) reaction, Protein A binds with lower avidity to the Fab region of certain immunoglobulin classes. The type of interaction of Protein A with immunoglobulins has two main practical advantages: first, since the antigen-binding region of the antibody molecule is not involved, any antigen-antibody reaction is unaffected, and second, because Protein A reacts with immunoglobulin from several different species, it has wide applicability in immunoassays.
While Protein A has found wide application in immunochemistry and immunohistochemistry, it has heretofore not been used as an internal assay control in immunoassay devices. This is probably because researchers believe that the Fc portion of the IgG in the sample to be assayed would bind the Protein A control, leaving only the Fab portion of the IgG exposed. Because the Fab portion of IgG does not bind to Protein A, one would not expect the system to work as an internal control. Thus, Protein A, although inexpensive and widely available, has not previously been used as a control substance in immunoassays devices.
The use of Protein A in immunoassay devices is described in U.S. Pat. No. 4,169,138, incorporated herein by reference, which discloses a method for detecting analyte antibodies bound to an antigen wherein a sample suspected of containing analyte antibody is added to a surface having antigen coated thereon. Next, Protein A which is bound to small water-insoluble particles is added. The particle/Protein A complex binds to analyte antibody, if present, thus forming a visible coating if analyte antibody has bound to the antigen.
U.S. Pat. No. 3,966,898, incorporated herein by reference, describes methods for labeling Protein A. The labeled Protein A is then used to detect IgG bound to an antigen or hapten.
None of the foregoing references is believed to disclose the present invention as claimed and is not presumed to be prior art. The references are offered for the purpose of background information.
There is an ongoing need to reduce the costs of immunoassay devices while improving upon their sensitivity and reproducibility. The antibodies and/or antigens typically used for assay controls can often be costly because their production and purification is labor-intensive. Another drawback of using antibodies for controls is that their sensitivity can vary from lot to lot, thus they can require substantial quality control testing. It would be desirable to have an internal control that, like antibody to human IgG, has the capability of binding to antibodies in human serum, yet is inexpensive to manufacture, reproducible, and comes from a reliable source.